Conclusions:%20There%20are%20some%20similarities%20but%20significant%20differences%20in%20the%20gene%20expression%20profile%20of%20cultured%20adult%20and%20immortalized%20ARPE%20cells,%20and%20it%20is%20important%20to%20note%20that%20some%20specific%20genes%20are%20only%20expressed%20in%20one%20of%20these%20two%20groups.%20These

1
DIFFERENCES IN GENE EXPRESSION PROFILE OF
CULTURED ADULT VERSUS IMMORTALIZED HUMAN RPE Lee
Geng, Hui Cai and Lucian V. Del Priore Department
of Ophthalmology, Columbia University, New York,
New York
Genes expressed in ARPE cells but not detected
in adult RPE cells
Purpose Immortalized human RPE (cell line
ARPE-19) are used widely to draw inferences about
the behavior of adult RPE. We have used DNA
microarray analysis to compare the gene
expression profiles of these two cell types.
A.
Methods Cultured primary RPE from five human
donors (age 48 - 80 years) and ARPE-19 cultured
to confluence in five dishes were used for DNA
microarray study. Total RNA was isolated using a
Qiagen RNeasy Mini Kit. First and second strand
cDNA were synthesized with a T7-(dT)24 oligomer
for priming and double-stranded cDNA was cleaned
with Phase Lock Gels-Phenol/Chloroform extraction
and ethanol precipitation. Biotin-labeled
antisense cRNA was produced by an in vitro
transcription reaction (ENZO BioArray High Yield
RNA Transcript Labeling Kit) and incubated with
fragmentation buffer (Tris-acetate, KOAc and
MgOAcat 94oC for 35 minutes). Target
hybridization, washing, staining and scanning
probe arrays were done following an Affymetrix
GeneChip Expression Analysis Manual. Microarray
data were treated with normalization, log
transformation, statistic determination for
presence or absence for RPE gene expression
profile. Clustering analysis, including PCA was
used with Affymetrix Microarray Suite 5.0,
Genesis 1.30 software.
B.
Genes expressed in adult RPE cells but not
detected in aRPE-19 cells
Figure 2. A. a scatter plot of expression levels
of about 6,000 genes in pRPE vs. aRPE-19 shows an
incomplete overlap in the gene expression
profiles of these two cells types. B. Figure
shows the distribution of differentially
(1.5-fold) expressed genes in pRPE and aRPE-19
cell types
Results
ARPE
Figure 3. The expression of 5,932 genes (out of
12,600 genes on microarray Human 95UA chip) was
detected in ARPE-19 cells, in comparison to
expression of only 4,849 genes in adult RPE cells
from all 5 human donor eyes aRPE-19 express
primary RPE
A.
B.
Conclusions There are some similarities but
significant differences in the gene expression
profile of cultured adult and immortalized ARPE
cells, and it is important to note that some
specific genes are only expressed in one of these
two groups. These studies suggest caution should
be exercised when generalizing results obtained
from ARPE-19 to results that would be obtained
with adult RPE.
Figure 1. Principle Component Analysis (A) and
hierarchic clustering analysis (B) demonstrate
that the gene expression profile of the adult RPE
(shown in blue color group) and ARPE-19 ( shown
in red color group) cluster into two distinct
groups with no discernable overlap.
Supported by Research to Prevent Blindness,
Robert L. Burch III Fund, and the Foundation
Fighting Blindness
2
AGING OF BRUCHS MEMBRANE DECREASES RETINAL
PIGMENT EPITHELIUM (RPE) PHAGOCYTOSIS Reiko
Koyama, Hui Cai and Lucian V. Del
Priore Department of Ophthalmology, Columbia
University, NY, New York
Purpose The earliest changes in age-related
macular degeneration occur within Bruchs
membrane. Bruchs membrane aging affects the
attachment and survival of the overlying RPE.1-3
Herein we determine the effects of Bruchs
membrane aging on RPE phagocytosis ability.
plt0.02
Methods Explants of human Bruchs membrane were
prepared as previously described. Donor
ages(young eyes ages 33 - 44 older group 73
-94). 1-3 Native RPE were removed by bathing the
choroid-BM-RPE complex with 0.02 N ammonium
hydroxide followed by washing in PBS. The
choroid-BM complex was set on polytetrafluoroethyl
ene membrane with the basal lamina of the RPE
facing the membrane. 4 agarose was poured onto
the choroid -BM- complex from choroidal side and
the tissue was kept at 4oC to solidify the
agarose. The polytetrafluoroethylene membrane
was peeled away and 6 mm circular buttons were
trephined from choroid-BM-gel complex. Buttons
were placed on 4 agarose at 37oC in non-treated
polystyrene wells of a 96 well plate (Figure1).
50,000 immortalized ARPE-19 were seeded onto
wells containing Bruchs membrane explants and
bare control wells (plastic only) for 72 hours(5
wells for each age group). 1ul of fluorescent
latex beads (3.6x105beads/ul) were added to each
well for another 24 hours. ARPE-19 were passaged
by trypsinization. Ingested beads were counted
using a FACS Flow Cytometer. Data were generated
with at least three independent experiments.
pgt0.05
of RPE ingesting beads
of RPE ingesting beads

Fig.3 Percentage of RPE cells that ingest beads
was higher for RPE cultured onto Bruchs membrane
explant than on bare plastic wells (22.2211.59
vs. 10.043.52).
Fig.4 Percentage of RPE cells which have the
ability to ingest beads on younger Bruchs
membrane versus older Bruchs membrane were
similar (25.7415.94 vs. 22.2211.59).
plt0.0003
plt0.008
Results
Flourescent intensity/cell
Fluorescent intensity/cell
A. on younger Bruchs membrane
B. on older Bruchs membrane
cells not ingesting beads
cells not ingesting beads
cells ingesting beads
cells ingesting beads
cell counts
cell counts
Fig. 5. Comparison of the phagocytosis ability of
RPE cells cultured on young Bruchs membrane
explant versus older BM. Average fluorescent
intensity per cell (measure of capacity of
phagocytosis per cell) on Bruchs membrane was
higher than on plastic (291.6180.91 vs.
167.5035.01).
Fig. 6 Observation of the population of cells
that had ingested beads. The average fluorescent
intensity per cell, which is a measure of
capacity of phagocytosis per cell in the younger
Bruchs membrane group was higher than on older
Bruchs membrane (312.6083.80 vs. 26771.08)
fluorescent intensity
fluorescent intensity
Fig.2. Flow cytometry histogram demonstrating the
distribution of fluorescent intensity. RPE cell
population which ingest fluorescent beads (green
zone) in young BM group (A) is larger than older
BM group (B).
Fig 1. Photo shows 6-mm circular buttons which
were trephined from BM and placed into 96 well
plate
Conclusions This study suggests that Bruchs
membrane promote RPE phagocytosis compared to
bare plastic tissue culture wells, and aging of
Bruchs membrane reduces the ability of RPE to
ingest beads. To our knowledge, this is the first
demonstration that aging of Bruchs membrane can
modulate RPE phagocytosis ability. Further study
is required to determine the implications of this
age-dependent decrease in RPE phagocytosis in the
pathogenesis of AMD.
References 1. Tezel TH, Del Priore LV, Kaplan
HJ. Fate of Human Retinal Pigment Epithelial
Cells Seeded onto Layers of Human Bruchs
Membrane. Invest Ophthalmol Vis Sci
199940467-476. 2. Tezel TH, Del Priore LV.
Repopulation of Different Layers of Host Human
Bruchs Membrane by Retinal Pigment Epithelial
Cell Grafts. Invest Ophthalmol Vis Sci
199940767-774. 3. Del Priore LV, Tezel TH.
Reattachment Rate of Human Retinal Pigment
Epithelium to Layers of Human Bruchs Membrane.
Arch Ophthalmol 116335-341, 1998. Supported by
Research to Prevent Blindness, Robert L. Burch
III Fund, and the Foundation Fighting Blindness
Supported by Research to Prevent Blindness,
Robert L. Burch III Fund, and the Foundation
Fighting Blindness.
3
BRUCH'S MEMBRANE AGING ALTERS THE RPE EXPRESSION
PROFILE OF PROLIFERATION, MIGRATION AND APOPTOSIS
BUT NOT ANGIOGENESIS GENES Hui Cai, Lucian V.
Del Priore Department of Ophthalmology, Columbia
University, New York, New York
B.
A.
PROLIFERATION GENES
OVERALL EXPRESSION
Purpose Principal component analysis (PCA) is a
technique used to determine global changes in
gene expression in response to changing cellular
conditions. We have used PCA to determine the
gene expression pattern of the retinal pigment
epithelium (RPE) in response to age-related
changes within Bruchs membrane (BM).
YOUNG BM OLD BM
PC2 22.0
PC2 18.3
Methods Immortalized human ARPE-19 cells were
seeded onto human BM (five young samples donor
age 31- 47 yr and five older samples donor age
71 81 years) harvested from human eye bank
eyes. ARPE-19 were harvested 72 hours after
seeding onto human BM and total RNA was isolated
using a Qiagen RNeasy Mini Kit. First and second
strand cDNAs synthesis, Biotin-labeled antisense
cRNA Target hybridization, washing, staining and
scanning probe arrays were done following an
Affymetrix GeneChip Expression Analysis Manual.
RPE gene expression profile was analyzed with
Affymetrix Miroarray Suite 5.0, SAM and Genesis
1.3 software.
PC3 12.4
PC3 10.7
PC1
47.2
PC1
27.4
C.
D.
MIGRATION GENES
ANGIOGENESIS GENES
PC2 21.0
PC2 18.4
Results
PC3 11.3
PC3 15.5
PC1 35/2
PC1
33.1
Figure 2. Principal component analysis of gene
expression. (A) The pattern of gene expression of
human RPE seeded onto Bruchs membrane explants
from younger donors (blue) shows a tighter
clustering than the gene expression profile of
RPE seeded onto older donors (red) Bruchs
membrane (B) Cell proliferation and migration
genes of RPE seeded onto young Bruchs membrane
(blue) show a tight clustering with spread in the
expression profile of proliferation-related genes
of human RPE seeded onto older Bruchs membrane
explants (red). (C) There was a similar pattern
for apoptosis-related and genes . (D) There is no
age-dependent alteration in the spread in the
expression profile of angiogenesis genes.
Figure 1. The expression of approximately 6,000
genes (out of 12,600 genes on microarray Human
95UA chip) was detected. Scatter plot of gene
expression within RPE cultured onto 31 year-old
vs 38 year-old Bruchs membrane. More than 96 of
genes are expressed consistently among all
samples tested within the young age group (data
not shown). The correlation co-efficient is 0.989
suggesting limited variation between these
individuals.
Table I. 20 genes and ESTs with the lowest
p-values. Microarray data suggests that aging of
Bruchs membrane increases the expression level
of numerous genes. We performed RT-PCR on several
genes of interest, including up regulated genes
transforming growth factor alpha, CD74 antigen,
and heparin-binding growth factor binding
protein, and down regulated genes that include
the ATP-binding cassette, vitronectin, cartilage
GP-39 protein, doublecortin and CaM kinase-like
1, and catenin. RT-PCR confirms the up regulation
of TGF alpha and the down regulation of
vitronectin, doublecortin and CaM kinase-like 1,
and Rho-related BTB domain containing 1.
Conclusions Age-related changes within BM alone
induce significant spreading of the gene
expression profile of proliferation, apoptosis
and cell migration genes, with no change in
angiogenesis genes. These observations suggest
some of cellular changes that develop within the
RPE as a function of age, such as occur in
age-related macular degeneration, may be the
result of substrate-induced alterations in the
behavior of the overlying RPE.
Supported by Research to Prevent Blindness,
Robert L. Burch III Fund, and the Foundation
Fighting Blindness
4
RPE EXPRESSION OF VITRONECTIN AND ITS RECEPTOR
ARE DOWNREGULATED WITH AGING OF HUMAN BRUCHS
MEMBRANE L. V. Del Priore, H. Cai, and T. H.
Tezel Department of Ophthalmology, Columbia
University, New York, New York
Purpose Previous studies shows that vitronectin
is a major constituent of ocular drusen and
vitronectin mRNA is synthesized in RPE cells. The
purpose of this study is to determine if Bruchs
membrane aging alters the level of vitronectin
mRNA and its receptor in the overlying RPE.
Figure 2. DNA microarray data show vitronectin
(VN) and its receptor alphaV transcript
expression patterns. ARPE 19 cells were cultured
on different aged Bruchs membrane explants for
72 hours. Data show VN and its receptor subunit
alphaV expression level decreases in RPE cells
overlaying on aged Bruchs membrane.
Methods DNA microarray and semi-quantitative
RTPCR method were used for this study.
Immortalized human ARPE-19 cells were seeded onto
human Bruchs membrane (five samples from donors
age lt 50 yr and five samples from donors age gt 70
yr) harvested from eye bank eyes. ARPE-19 cells
were harvested 72 hours after seeding onto human
Bruchs membrane and total RNA was isolated using
a Qiagen RNeasy Mini Kit. First and second
strand cDNAs synthesis, Biotin-labeled antisense
cRNA target hybridization (on Affymetrix U95A
chip), washing, staining and scanning probe
arrays were done following an Affymetrix GeneChip
Expression Analysis Manual. Real-time
quantitative polymerase chain reaction (Roche
Lightcycler) using samples generated from
different experiments with different donor
tissues were used to confirm and further study
the genes expression patterns. Oligo primers were
determined with LC Primer Design and RTPCR data
were analyzed with Lightcycler3 Data Analysis
software.
GADPH
VN
B.
A.
Results
Figure 3. Real time semi-quantitative RT-PCR. The
Bruchs membrane samples were from different
batches of donors than those shown above. (A)
Quantitative RT-PCR was performed , after
establishing standard curves with GAPDH
house-keeping gene using serial dilutions of
total RNA. (B) Vitronectin expression level is
decreased in RPE cells seeded onto aged Bruchs
membrane (dashed lines in duplicates). (C)
vitronectin receptor alphaV subunit mRNA in RPE
cells is also decreased upon culturing on aged BM
(dashed lines).
C.
VN RECEPTOR
Figure 1. A. Plot of individual DNA microarray
data on vitronectin (in red color) and its
receptor subunit alphaV (in blue color)
transcript expression levels. Data show a general
decreased expression level trends for both VN and
its receptor mRNA in RPE cells seeded onto aged
Bruchs membrane. B. Heat map shows VN and its
receptor expression levels (high level in red
color and low level in green) in RPE cells
cultured on BM explant from different donor ages.
Conclusions Aging of Bruchs membrane
downregulates the expression profile of
vitronectin mRNA and its receptor in human RPE.
The vitronectin receptor may play an important
role in phagocytosis of photoreceptor outer
segments and vitronectin partially mediates RPE
attachment to human Bruchs membrane. These
observations suggest some of the changes seen in
age-related macular degeneration may be the
result of substrate-induced alterations in the
behavior of the overlying RPE.
Supported by Research to Prevent Blindness,
Robert L. Burch III Fund, and the Foundation
Fighting Blindness.
5
ANATOMICAL SIMILARITIES BETWEEN STAGE 3 AND STAGE
4 MACULAR HOLES IMPLICATIONS FOR TREATMENT
Jon Wender, Tomohiro Iida, M.D., Lucian V. Del
Priore, M.D., Ph.D. Department of Ophthalmology,
Columbia University, New York, NY
ABSTRACT Purpose To determine whether the
observed anatomy of macular holes can be
explained by a hydrodynamic model in which fluid
flow through the hole is balanced by fluid
pumping across the RPE. We use this model to draw
conclusions about the possible role of
vitreomacular traction in determining the
morphology of macular holes and their resolution
after vitreous surgery. Design Cross sectional.
Methods Retrospective study in a clinical
practice. The study included 42 eyes of 42
patients, each with a stage 3 or 4 macular hole
(Gass classification). Macular holes were staged
on the basis of clinical exam, Optical Coherence
Tomography, and intraoperative findings. We
measured the radius of the macular hole and the
radius of the surrounding cuff of subretinal
fluid from color or red free fundus photographs,
and determined the relationship between these 2
variables. Results The mean age of the patients
was 68.0 ? 7 years old (range 51-80). 25 patients
had stage 3 macular holes and 17 patients with
stage 4 macular holes. The radius of the
neurosensory detachment radius was related to the
square of the macular hole radius for stage 3 and
stage 4 holes, with no significant difference
between the stage 3 and stage 4 linear trend
lines (p0.999). There was no correlation between
patient age and the area of the macular hole (r
0.0645) or neurosensory detachment plus hole
(r0.156) over the range of age in this study
(51-80 years). However, the area of the
doughnut-shaped cuff of subretinal fluid
increased with increasing patient age (p
0.0493), thus suggesting an age-dependent decline
in the pumping ability of the RPE. Conclusions
Our data is consistent with a hydrodynamic model
in which macular hole anatomy is determined by a
balance between fluid flow through the hole and
fluid outflow across the RPE. Since Stage 3 and 4
macular holes exhibit a similar relationship
between the size of the macular hole and the size
of the cuff of subretinal fluid around the hole,
simple relief of vitreomacular traction would not
lead to resolution of the subretinal fluid cuff
unless it is accompanied by a reduction in hole
diameter due to approximation of wound edges.
Results

• Mean age 68.0 ? 7 years (range 51-80)
• 25 patients with stage 3 macular holes
• 17 patients with stage 4 macular holes
• The neurosensory detachment radius was related
  to the square of the macular hole radius for
  stage 3 and stage 4 holes, with no significant
  difference between the stage 3 and stage 4 linear
  trend lines (p0.999).
• Similarly, the neurosensory detachment area was
  related to the square of the macular hole area
  for stage 3 and stage 4 holes, with no
  significant difference between the stage 3 and
  stage 4 linear trend lines.

Mathematical Model of Macular Holes Our model
relies on the fact that there is flow of fluid
from the vitreous cavity, across the intact
retina and RPE, into the choroid in the normal
human eye. The development of a full thickness
defect in the neurosensory retina will allow
fluid from the vitreous cavity to flow through
the hole and detach the retina from the RPE (Fig.
1). Fluid flow through the hole will create an
enlarging neurosensory detachment. The
neurosensory detachment around the hole will
increase in size and ultimately be limited by the
pumping ability of the underlying RPE. In
equilibrium (i.e., when there is no further
enlargement of the hole), the fluid flow into the
hole (Fin) and fluid flow out of the hole through
the RPE (Fout) will be equal (Fig. 1). For a
Newtonian fluid, the rate of fluid flow into the
hole is limited by the size of the macular hole
itself. Mathematically, fluid flow through the
macular hole (Fin) is inversely proportional to
the resistance (R) to fluid flow through the
hole. Thus, we write (Eq. 1) Fin ? 1/R The
resistance is proportional to the square of the
area of the macular hole, where the area of the
macular hole is given by ?r12. Thus, (Eq.
2) Fin ? / 1/?2r14 ??2r14, where ? is an
arbitrary constant. The flow out (Fout) of the
subretinal space is due to active pumping of
fluid by the underlying RPE. If we assume that
the pumping ability of the RPE is homogeneous
(i.e., does not vary across the area of the
neurosensory detachment), then outward flow will
be directly proportional to the area of the RPE
under the macular hole and the surrounding
neurosensory detachment. Thus, we write (Eq.
3) Fout K?r22 where r2 is the radius of the
surrounding neurosensory detachment (Fig. 1). A
priori, it is not known if K is a constant or
varies with patient age. We note this explicitly
by writing (Eq. 4) Fout K(age) ?r22 In this
model, the subretinal fluid cuff will increase in
size until enough RPE is exposed to allow the
flow out to balance the fluid inflow through the
hole. In equilibrium, (Eq. 5) Fin
Fout (Eq. 6) ??2 r14 K?r22 (Eq. 7) r14
(K/??) r22 (Eq. 8) r12 ?(K/??) r2 Thus, the
hydrodynamic model predicts that r12 would be
proportional to r2 i.e., as the radius of the
macular hole doubles, the radius of the
neurosensory detachment would quadruple.
Figure 4. Subretinal fluid cuff area (a2-a1) vs.
macular hole area (a1) for stage 3 and stage 4
macular holes. There appears to be no
significant difference between the 2nd order
polynomial trend lines for stage 3 (y 10-05x2 -
0.7656x 258038, R2 0.6752) vs. stage 4 (y
10-05x2 - 1.8772x 426462, R2 0.8225) macular
holes.
Figure 2. Relationship between neurosensory
detachment radius (r2) and macular hole radius
squared (r12). Data fit to a linear regression
model, with no significant difference between the
stage 3 (y 0.0042x 220.04, R2 0.8098) and
stage 4 (y 0.0042x 243.61, R2 0.8046)
linear trend lines (p0.999).
FIGURE 1. (Top) Schematic diagram of fluid
dynamics of macular hole with surrounding
neurosensory detachment. r1 radius of macular
hole, r2 radius of subretinal fluid cuff.
(Bottom) Diagram illustrating fluid dynamics for
macular holes. Fin and Fout represent the fluid
flow into and out of the subretinal space,
respectively. For a Newtonian fluid, Fin is
inversely proportional to the resistance. Fout is
proportional to the area of the underlying RPE.
Figure 3. Relationship between neurosensory
detachment area and macular hole area squared.
Note that the data now fits a linear regression
model with no significant difference noted
between stage 3 (y 10-05 275658, R2 0.7677)
and stage 4 (y 10-05x 337788, R2 0.8691)
linear trend lines (p0.904).
Conclusions Our data is consistent with a
hydrodynamic model of macular hole anatomy in
which fluid flow through the hole is balanced by
the outflow of fluid across the RPE. Since Stage
3 and 4 macular holes exhibit a similar
relationship between the size of the macular hole
and the size of the cuff of subretinal fluid
around the hole, simple relief of vitreomacular
traction would not lead to resolution of the
subretinal fluid cuff unless it is accompanied by
a reduction in hole diameter due to approximation
of wound edges.
Methods Retrospective study in a clinical
practice. The study included 42 eyes of 42
patients, each with a stage 3 or 4 macular hole
(Gass classification). Macular holes were staged
on the basis of clinical exam, Optical Coherence
Tomography, and intraoperative findings. We
measured the radius of the macular hole and the
radius of the surrounding cuff of subretinal
fluid from color or red free fundus photographs,
and determined the relationship between these 2
variables.